Tension Induces a Base-Paired Overstretched DNA Conformation

Abstract

Type II topoisomerases maintain DNA topology by regulating the level of supercoiling of chromosomes. DNA gyrase is a unique and highly conserved bacterial Type II topoisomerase which is able to introduce negative supercoils into the genome. Using magnetic-tweezers, we assessed the supercoil generation and relaxation activities of Escherichia coli and Salmonella typhimurium DNA gyrases. Our results indicate that under single-enzyme conditions and 0.6-pN tension, both enzymes relax DNA at similar rates, but Salmonella gyrase pauses more often. At high enzyme concentration and lower tensions, Salmonella gyrase introduced negative supercoils faster and to a larger extent than E. coli gyrase. Sequence and structure analyses show that most of the differences between the two enzymes are in the C-terminal domain, involved in DNA wrapping. Our ongoing single-molecule experiments to assess the DNA wrapping activities of both enzymes may reveal differences in their mechanistic properties.

Nanomechanics of Diaminopurine-Substituted DNA

“…Simulations have shown that whereas G-C pairs persist through the transition, A-T pairs often form melted bubbles (30,37). Similarly, various pulling experiments and theoretical models have also demonstrated that at low-to-moderate monovalent salt conditions (<150 mM), AT-rich sequences tend to melt more easily than GC-rich sequences (24,28,29,(65)(66)(67)(68)(69), whereas under high-salt conditions interstrand charge repulsion is reduced, and AT-rich sequences also form S-DNA (31,32). Moreover, cyclic pulling experiments demonstrate that force-induced melting is associated with hysteresis, such that once melted, the complementary strands reanneal slowly (21,28,32,70).…”

“…For WT-DNA in physiological salt conditions, the characteristic overstretching transition around 60 pN is caused by a combination of factors, including force-induced melting (through strand peeling and melting bubble formation) and adoption of the S-DNA conformation (24,32,65,70,80). Several studies have shown that forceinduced melting tends to originate at AT-rich regions (28)(29)(30)37). In particular, a detailed study conducted by Bosaeus et al comparing the overstretching behavior of short 70% AT and 60% GC oligonucleotides (oligos) found that whereas AT sequences melt and GC sequences form S-DNA, the transition threshold for GC oligos was similar if not slightly higher than AT oligos: 63.3 5 1.1 pN for GC versus 61.5 5 2.6 pN for AT (28,29).…”

“…The pathway by which DNA becomes overstretched is a function of local stability and is determined by buffer conditions and sequence (26,27). Experiments using relatively short sequences have demonstrated that in physiological salt conditions, AT-rich regions tend to denature upon overstretching, whereas GC-rich regions remain hybridized (28,29). Similarly, molecular dynamic simulations suggest that stronger base pairing in GC-rich regions maintains base registration and minimizes interstrand separation, producing a higher transition force (30).…”

Structural Studies on Porphyrin–PNA Conjugates in Parallel PNA:PNA Duplexes: Effect of Stacking Interactions on Helicity

“…The hypothesis for a switch from a helical form to a ladder‐type structure has also been proposed for the transition occurring in DNA in stretching experiments at about 60–70 picoNewton (pN) of applied force, for which a ladder‐type structure (named S‐DNA) was proposed on the basis of experimental data and molecular dynamics (MD) calculations . Although the existence of this hypothetical structure has been subsequently questioned by other authors, on the basis of an alternative model of partial denaturation under tension, hydrogen‐bonded overstretched states of DNA have been found to be formed for GC‐rich sequences, where they probably play an important role in some biological processes such as in DNA recombination …”

“…58 Although the existence of this hypothetical structure has been subsequently questioned by other authors, on the basis of an alternative model of partial denaturation under tension, 59,60 hydrogen-bonded overstretched states of DNA have been found to be formed for GC-rich sequences, where they probably play an important role in some biological processes such as in DNA recombination. 61 Interestingly, X-ray diffraction or NMR experiments by other authors have pointed out as well that PNA can adopt a broad range of structural motifs in duplexes. 62,63 For example, a modulation of the PNA:PNA helicity was demonstrated by Renikuntla and Armitage.…”